The objective of this prospective study was to compare the number of CIN2+cases detected in negative cytology by different quality control (QC) methods. Full rescreening, high-risk (HR) human papillomavirus (HPV)-targeted reviewing and HR HPV detection were compared. Randomly selected negative cytology detected by BD FocalPoint (NFR), by guided screening of the prescreened which needed further review (GS) and by manual screening (MS) was used. A 3-year follow-up period was available. Full rescreening of cytology only detected 23.5% of CIN2+ cases, whereas the cytological rescreening of oncogenic positive slides (high-risk HPV-targeted reviewing) detected 7 of 17 CIN2+ cases (41.2%). Quantitative real-time PCR for 15 oncogenic HPV types detected all CIN2+ cases. Relative sensitivity to detect histological CIN2+ was 0.24 for full rescreening, 0.41 for HR-targeted reviewing and 1.00 for HR HPV detection. In more than half of the reviewed negative cytological preparations associated with histological CIN2+cases no morphologically abnormal cells were detected despite a positive HPV test. The visual cut-off for the detection of abnormal cytology was established at 6.5 HR HPV copies/cell. High-risk HPV detection has a higher yield for detection of CIN2+ cases as compared to manual screening followed by 5% full review, or compared to targeted reviewing of smears positive for oncogenic HPV types, and show diagnostic properties that support its use as a QC procedure in cytologic laboratories.